ABSTRACT Targeting therapeutic biomolecules to diseased cells and tissues remains a major hurdle in the development of drug delivery methods. An important potential advance is the recent utilization of engineered extracellular vesicles (EVs), which include both exosomes and microvesicles, each with surface-displayed tumor targeting motifs. This novel therapeutic modality has shown the ability to deliver nucleic acid-based therapeutics to a variety of cell types, including various cancers, and has a demonstrated success with human hepatocellular carcinoma cells. Unfortunately, the isolation and purification of engineered therapeutic EVs remains a laborious and expensive task, and is a significant obstacle to the clinical development of these methods. Current approaches rely on ultra-centrifugation or precipitation, but because conventional biopharmaceutical production cells produce many types of EVs, the purified product is typically dominated by empty or off-target EVs and other particulates. These contaminants may be immunogenic and can produce undesirable side effects, while also decreasing the potency of the EV therapeutic. Consequently, there is an urgent need to develop a robust purification scheme that enriches for engineered EVs over other contaminants. We propose to address this challenge through a novel application of a modular, self-cleaving purification tag that we have developed for recombinant protein purification in our previous work. In this system, a 36 amino acid peptide segment derived from a self-cleaving intein is appended to the tumor-targeting protein on the surface of an engineered EV. This intein segment acts as a dock for a complimentary intein segment, which is produced in a separate process and is fused to any of a number of purification tags. Once the EVs are expressed, the intein- fused purification tag is added and associates strongly with the surface of the EV through association of the intein segments. Depending on the intein-fused tag, a highly selective purification of the EV becomes possible. Once purified, the entire assembled intein and tag is induced to self-cleave from the surface of the EV, leaving the native and tagless targeting protein on the EV surface. Importantly, because the purification method is encoded by the complimentary intein segment, which can be produced in a separate process, the EV does not have to be re-engineered for each new purification strategy. In this work, we propose to develop this strategy using a previously engineered model EV particle to display the 36 amino acid intein segment on its surface. The EV particles will initially be produced by transient transfection in HEK293 cells, with the expectation that this method can ultimately be extended to stable biopharmaceutical production cell lines. We will examine monolithic chromatographic supports to develop and evaluate these methods, since they are commonly used to purify viruses and virus-like-particles (VLPs). Additionally, we will use selective precipitation and conventional affinity methods, enabled by the highly modular design of the proposed method. Evaluation of the intein fusion impacts and purified EVs will be based on western blots, qRT-PCR and Nanosight techniques.